A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for
maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple
factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL
conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs,
a comprehensive set of tools for studying modifications in Drosophila and mammals, based on
multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the
bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single
vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing
conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and nonspecific
background. We demonstrate the utility of the method in Drosophila cells and transgenic flies,
identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we
show conjugation and localization for many different UbLs, with the identification of novel potential
substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL
system a powerful complement to existing strategies for studying this important mode of protein
regulation